Field of Invention
This invention relates to the treatment of infectious diseases, certain heterocyclic compounds useful as therapeutic agents therein and, to pharmaceutical formulations containing said therapeutic agents. In particular this invention relates to a method for treating Mycobacterium tuberculosis infections in man, certain carboxy substituted 2-aryloxazoline, 2-arylthiazoline, 2-arylimidazoline compounds and similarly substituted related unsaturated heterocyclic compounds e.g. oxazole, thiazole and imidazole compounds, and to pharmaceutical formulations comprising said heterocyclic compounds.
Among bacterial infections, tuberculosis is usually a respiratory infection, although it can cause damage to virtually any organ in the human body. It is estimated that about 40% of the world""s population is infected with the tuberculosis bacterium. Early in this decade, the incidence of tuberculosis began rising after a 33-year downward trend. Streptomycin, the first antibiotic capable of killing the tuberculosis organism, was discovered in 1944. Other antituberculosis agents were subsequently developed. (Lemke, T. L. In Principles of Medicinal Chemistry, 4th ed.; Foye, W. O., Lemke, T. L. Williams and Wilkins: Baltimore, 1995; pp 747-758.) However, the recent emergence of drug resistant strains of tuberculosis has raised serious concern. Between 1989 and 1992 drug resistant strains had appeared in 17 states. (Tuberculosis and HIV Public Health Policy: A Dual Challenge. Washington: AIDS Action Foundation, March 1992). Recently, studies on the application of siderophore substituted mycobactins and analogs thereof were reported (Xu, Yanping, and Miller, Marvin J. J. Org. Chem., 1998,63, 4314). It was found that structural variations of the natural mycobactins produced by the mycobacterium were effective inhibitors of the mycobacterium. The retrograde synthesis work on the mycobactins carried out in this study has led to the discovery of the antituberculosis therapeutic agents of this invention.
The compounds provided by this invention are useful in the method described herein for the treatment of tuberculosis and are represented by the following formula 1. 
wherein X is O, S, or NH, 
Y represents fragments
Y1 is OR4, wherein R4 is hydrogen, C1-C4 alkyl, phenyl, benzyl, diphenylmethyl, or a biologically-labile ester, xe2x80x94NHR4xe2x80x2, or xe2x80x94NHNHR4xe2x80x2 wherein R4xe2x80x2 is hydrogen, C1-C4 alkyl, phenyl, benzyl, diphenylmethyl, or a 5- or 6-membered saturated or unsaturated heterocycle containing from 1 to 3 of the same or different nitrogen, oxygen or sulfur hetero atoms, and wherein said phenyl, benzyl, diphenylmethyl and R4xe2x80x2 groups can be substituted by R as defined herein;
R is hydrogen or a substituent group e.g. lower alkyl, halogen, alkoxy, amino, substituted amino, hydroxy, carboxy or aminocarbonyl;
R1 is hydrogen, alkyl, 5- to 7- membered cycloalkyl, alkyl substituted by phenyl or diphenyl, e.g. benzyl or diphenylmethyl, or a t-vinyl or ethinyl group represented by the formulas xe2x80x94C(C1-C3)2xe2x80x94CHxe2x95x90CH2, xe2x80x94C(C1-C3)2xe2x80x94Cxe2x89xa1CH;
R2 is hydrogen or C1-C3 alkyl; and
R3 is hydrogen or lower alkyl;
The compounds represented by the formula 1 inhibit the growth of Mycobacterium tuberculosis and are useful in the treatment of tuberculosis in man when administered in an effective non toxic amount. Pharmaceutical formulations comprising a compound of the formula 1 or a pharmaceutically acceptable salt thereof and a suitable carrier are useful in the method for treating tuberculosis.
The terms used in the above formula 1 have the following meanings herein
R is hydrogen, C1-C4 alkyl such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, or t-butyl; C1-C4 alkoxy such as methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy and the like; C1-C4 alkoxy substituted by phenyl, diphenyl such as benzyloxy diphenylmethoxy, 2-phenylethoxy, 1,2-diphenylethoxy, 3-phenylpropoxy, 2,2-diphenylethoxy, and the like; halogen such as fluoro, chloro, or bromo; hydroxy; carboxy; cyano; aminocarbonyl; amino, mono- or di-C1-C4 alkyl)aminocarbonyl such as dimethylaminocarbonyl, methylaminocarbonyl, ethylaminocarbonyl, diethylaminocarbonyl, iso-propylaminocarbonyl, n-propylaminocarbonyl, t-butylaminocarbonyl, and the like; amino and mono- or di-C1-C4 alkyl)amino such as methylamino, ethylamino, t-butylamino, dimethylamino, diethylamino, methylethylamino, n-propylamino, and the like;
R1 is hydrogen, C1-C4 alkyl as exemplified for the term R above; 5- to 7-membered cycloalkyl such as cyclopentyl, cyclohexyl, cycloheptyl and the like; a di-C1-C3 alkyl) carbinyl ethinyl or vinyl-t-carbinyl group of the formula xe2x80x94C(C1-C3)2CHxe2x95x90CH2 or xe2x80x94C(C1-C3)2xe2x80x94CHxe2x95x90CH2 such as dimethylethinylcarbinyl, dimethylvinylcarbinyl, methylethylethinylcarbinyl, diethylethinylcarbinyl and the like; C1-C4 alkyl substituted by phenyl or diphenyl such as benzyl, diphenylmethyl, 2-phenylethyl, 2,2-diphenylethyl, 1,3-diphenylpropyl and, wherein said phenyl and diphenyl groups can be substituted by a group R as defined herein above;
R2 is hydrogen or C1-C3 alkyl;
R3 is hydrogen or C1-C4 alkyl;
R4 is hydrogen, C1-C4 alkyl, phenyl, benzyl, diphenylmethyl or a biologically-labile ester;
R4xe2x80x2 hydrogen, C1-C4 alkyl, phenyl, benzyl, diphenylmethyl, or a 5- or 6-membered saturated or unsaturated heterocyclic ring containing from 1 to 3 of the same or different nitrogen, oxygen, or sulfur hetero atoms such as for example, pyridyl, piperidyl, pyrrolidyl, pyrrolidinyl, pyrimidyl, pyrazolyl, furyl, thienyl, thiazolyl, oxazolyl, thiazinyl or tetrazolyl, and wherein said phenyl, benzyl, diphenylmethyl and heterocyclic ring groups can be substituted by R substituents having the same meanings as defined herein above.
When R4 is a biologically-labile ester, R4 represents an acyloxymethyl group, xe2x80x94CH2xe2x80x94C(O)-alk, a acyloxyalkyl group, alk-C(O)Oxe2x80x94CH-alk or alk-OC(O)Oxe2x80x94CH-alk, a alkoxyethyl ether group, alk-O-(CH2)2xe2x80x94Oxe2x80x94CH2xe2x80x94, where in the foregoing alk is C1-C4 alkyl, or is R4 phthalidyl, indanyl, or a 2-oxo-1,3-dioxolene group represented by the formula. 
Examples of such labile ester groups are acetoxymethyl, propionoxymethyl, pivaloyloxymethyl, 2-(methoxy)ethoxymethyl, 2-(ethoxy)ethoxymethyl, and 2-(t-butoxy)ethoxymethyl, and like labile ester moieties.
The compounds represented by the formula 1 having basic or acidic groups form salts with mineral acids and organic acids and inorganic and organic bases respectively. For example, when an R substituent is a basic group such as an amino or substituted amino group or when R4 is a basic heterocyclic group such as piperidyl or pyridyl, salts can be formed with mineral acids such as hydrochloric, hydrobromic, sulfuric and phosphoric acids. Organic acids which can be used are e.g. the sulfonic acids such as methanesulfonic, benzenesulfonic, toluenesulfonic, or naphthalenesulfonic; benzoic, chlorobenzoic, salicylic, malonic, maleic and succinic acids. Acidic groups e.g., when R is a carboxy group, form salts with basis such as sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium or potassium bicarbonate and ammonia. Also such acidic groups form salts with organic bases such as alkylamines e.g., methylamine, t-butylamine, diethylamine, cyclohexylamine, dicyclohexylamine, abietylamine, diethanolamine, and ethanolamine. Such salts are useful in the isolation and purification of formula 1 compounds and in the preparation of pharmaceutical formulations for administration in the therapeutic method provided herein.
Preferred compounds of the invention are represented by the formula 1 wherein Y is the fragment (1a) or (1b) and X is O or S. In particular, of the preferred compounds are those wherein R1 is benzyl or diphenylmethyl and Y1 is OR4 wherein R4 is benzyl or diphenylmethyl are preferred. Among this preferred group, the compound (S)-Benzyl 2-[2-(benzyloxy)phenyl]-2-oxazoline-4-carboxylate, in in vitro tests inhibited 98% of the growth of M. tuberculosis H37R at a concentration of 12.5 xcexcg/mL.
According to the method provided by this invention Mycobacterium tuberculosis infections in man are treated by administering to the infected host an effective, non toxic amount, of a compound represented by the formula 1 or a pharmaceutically acceptable non toxic salt thereof. An effective amount of the compound of formula 1, in general, is between about 2 mg/kg of host body weight and about 25 mg/kg. The compound of formula 1 or a pharmaceutically acceptable salt thereof may be administered in a single daily dose or in multiple doses throughout the day. The amount administered and the particular dose regimen employed by the physician may vary depending on such factors as the severity of the disease, the duration of the infection prior to treatment, the age and general health of the infected host and the tolerance of the particular host to the treatment. The compound represented by the formula 1 or a pharmaceutically acceptable salt thereof may be administered parenterally or orally in a suitable pharmaceutical formulation. The route of administration may be determined by the acceptance of the dose regime by the individual host. Parenteral administration is achieved via im. injection or via iv. For such administration the compound or a salt thereof is formulated in a suitable diluent such as physiological saline, glucose or dextrose solution or Ringer""s solution and the like. For iv. administration the drip method can be employed as well as the piggy back method wherein the drug formulation is added slowly to the host along with other fluids such as mineral solutions or nutrients such as glucose and amino acids. For oral administration a compound of the invention or a salt thereof can be formulated into gelatin capsules, e.g. in 250 mg and 500 mg doses, in tablet form or as a liquid solution or suspension.
The invention also provides pharmaceutical formulations for use in the above-described method. The formulations comprise a compound of the formula 1 or a pharmaceutically acceptable non toxic salt thereof and a pharmaceutically acceptable carrier. Suitable carriers for im. or iv. administration include for example, deionized sterile water, physiological saline, 5% glucose and like commonly use carriers. Tablets may be formulated with the drug and binding agents, solubilizing agents, fillers and the like. Liquid suspensions which may be used can comprise the drug and water, a flavoring agent, solubilizing agent, coloring agent, a preservative and the like.
The pharmaceutical formulations provided herein may be made up in unit dosage form. For example, a formulation comprising 100 mg of the compound or a salt thereof and a pharmaceutically acceptable carrier. Other unit dosage formulations comprise 1 g of the drug or salt form thereof and a pharmaceutically acceptable carrier or 500 mg of the compound of formula 1 or a salt thereof and a pharmaceutically acceptable carrier. For im. administration the unit dose may be incorporated in sterile ampoules or vials while for iv. administration the unit dose may be provided in sterile bottles adaptable to hook up with tubing for the drip method with controls for regulating the rate of flow.
The compounds of the invention represented by the formula 1 are prepared by synthetic routes which depend upon the heterocyclic ring desired where, in formula 1, X is O, S, or NH. The compounds where, in the formula 1, Y is the fragment (1a) and X is O (oxazolines) are prepared by coupling an ester of the amino acid ester, HOxe2x80x94CH(R3)xe2x80x94CH(NH2)xe2x80x94C(O)ester, e.g. an ester of serine or threonine, with a 2-(R1O)benzoic acid to provide the amide, N-[2-(R1O)benzoyl]-NHCHxe2x80x94[CH(R3)xe2x80x94OH]xe2x80x94C(O)ester The coupling reaction can be carried out via formation of an active ester of the acid such as that formed with 1-hydroxy-7-azabenzotriazole or via treatment of the acid and amine with a carbodiimide. The amide is then cyclized with Burgess"" reagent (methoxycarbonylsulfamoyltriethylammonium hydroxide inner salt) to yield the ester of 2-[2-(R1O)phenyl]-2-oxazoline-4-carboxylic acid. The preparation is illustrated with serine and threonine in the following reaction scheme 1. 
In Reaction Scheme 1 Bn represents benzyl.
The thiazoline compounds represented by the formula 1, X is S, wherein Y is the fragment (1a) are prepared in a similar manner. The amino acid ester is coupled with the acid to form the amide, N-[2-(R1O)benzoyl]-NHxe2x80x94CHxe2x80x94[CH(R3)xe2x80x94OH]xe2x80x94C(O)ester, and the amide is cyclized with Lawesson""s reagent (2,4-bis(p-methoxyphenyl)-1,3-dithiadiphosphetane-2,4-disulfide as shown with serine and threonine in the following Reaction Scheme 2. 
The imidazolidine compounds represented by the formula 1 where X is NH and Y is the fragment (1a) are prepared by coupling a 2-R1O substituted benzoic acid with a 2,3-diaminopropionic acid ester wherein the 3-amino group is protected to form the amide, N-[2-(R1O)benzoyl]xe2x80x94NHxe2x80x94CHxe2x80x94[CH(R3)xe2x80x94NH2]xe2x80x94C(O)ester, wherein the free amino group is protected. The coupling is carried out with an active ester of the carboxylic acid or with a carbodiimide. Following the coupling reaction the amino protecting group of the amide formed is removed to form the amino substituted amide, and the amino amide is cyclized to form the imidazolidine ring via dehydrative cyclization as depicted below. 
The 2,3-diaminopropionic acid esters, NH2xe2x80x94CHxe2x80x94[CH(R3)xe2x80x94NH2]xe2x80x94C(O)ester, are prepared as follows. t-Boc protected serine, HOCH2CH(NH-t-Boc)COOH, is coupled with O-benzylhydroxylamine at pH 4-5 with EDC HCl to provide the hydroxamate, HOCH2CH(NH-t-Boc)CONHOBn. The latter is cyclized in acetonitrile with triphenylphosphene-carbon tetrachloride in the presence of triethylamine to form the xcex2-lactam, 1-benzyloxy-3-t-Boc-aminoazetidin-2-one. The xcex2-lactam is then saponified with lithium hydroxide to provide, in 100% yield, the 2,3-diaminopropionic acid derivative, BnONHCH2CH(NH-t-Boc)COOH. In the above Bn is benzyl. The carboxylic acid group of the derivative obtained is esterified for use in the coupling reaction of Reaction scheme 3. The OBn protecting group is left intact during the coupling reaction while the t-Boc protecting group is removed to provide the free amino group involved in the coupling. Following amide formation the OBn group is removed via hydrogenation to provide the free amino amide ester for cyclization to the imidazoline. The above sequence of reactions is illustrated below. 
Compounds represented by the formula 1 where Y is the fragment (1a) and R2 is C1-C3 alkyl are obtained with the corresponding oxazoline, thiazoline or imidazoline compound e.g., a methyl or benzyl 2-[2-(benzyloxy)phenyl]-2-oxazoline-5-H, or 5-(C1-C4 alkyl)-4-carboxylate, by reaction at about xe2x88x9278xc2x0 C. in an inert solvent such as THF with a C1-C3 alkyl iodide in the presence of a base such as sodium dimethylsilylamide as depicted in the following. 
The oxazole, thiazole and imidzole compounds represented by the formula 1 wherein Y is the fragment (1b) are obtained by dehydrogenation of the corresponding oxazoline, thiazoline or imidazoline by treatment with a dehydrogenation reagent such as DDQ. The reaction is carried out at a temperature of about 50xc2x0 C. and about 75xc2x0 C. in an inert solvent such as benzene and is illustrated below. 
The compounds represented by the formula 1 wherein Y1 is xe2x80x94NHxe2x80x94R4xe2x80x2 are prepared by the reaction of an ester of (1a) or (1b), Y1xe2x95x90C(O)OR4, wherein OR4 is e.g. C1-C4 alkoxy or an acyl halide of the carboxy group, (Y1xe2x95x90C(O)OH, R4xe2x95x90H), with the NH2R4xe2x80x2 amine. The compounds wherein Y1 is xe2x80x94NHNHR4xe2x80x2 can be obtained by the reaction of the hydrazine H2NNHR4xe2x80x2 with the acyl halide or ester of the carboxy group. Compounds of the formula 1 wherein R4xe2x80x2 is a heterocycle e.g. pyridine, are prepared by reacting the acyl halide or the ester of Y1 with an amino substituted pyridine, e.g. 4-aminopyridine. The compounds wherein Y1 is xe2x80x94NHNHR4xe2x80x2 are obtained by reacting the heterocyclic hydrazine with the acid halide or the ester wherein, OR4 is e.g. an alkoxy group, to provide the corresponding amide or hydrazide. Examples of heterocyclic substituted compounds are represented by the following formulas wherein Het represents the heterocyclic ring. 
Het can be 2- or 4-pyridyl, 2- or 4-piperidyl, 3-thienyl, 2-(1,3-thiazolyl), 3-pyrimidyl, 2-oxazolyl, 1H-tetrazolyl and like heterocycles. The hydrazino compounds where in the above formulas xe2x80x94NHxe2x80x94Het is replaced by xe2x80x94NHNHxe2x80x94Het like heterocycles are represented by Het.
In carrying out the preparation of the compounds represented by the formula 1 as shown above, when any of the groups represented by R, R1 or R4 bear an amino or carboxylic acid substituent, these groups are best protected with a commonly used protecting group to prevent untoward side reactions caused by participation of the unprotected amino or carboxy groups. Amino protecting groups which may be used for temporary protection of an amino group include, among many, tert-butyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzylcarbonyl, trichloroacetyl, silyl groups such as trimethylsilyl, t-butyldiethylsilyl, t-butyldiphenylsilyl and the like, enamines such as those formed with ethyl acetoacetate or methyl acetoacetate, and like amino protecting groups. The carboxylic acid substituent can be protected with such groups as a silyl group such as those listed above, benzyl, p-nitrobenzyl, t-butyl, and the like. The particular group chosen will be one that is removed under conditions differing from the conditions employed in the desired reaction so that the protecting group will survive the desired reaction and thereafter be removed to obtain the product.
The following examples are provided to further illustrate the methods for preparing the compounds of the invention and are not to be considered as limitations of the invention.
In the examples the following abbreviation designations of reagents have the indicated meanings:
FABMSxe2x80x94fast atom bombardment mass spec.
HREIMSxe2x80x94high resolution electron impact mass spec.
DCCxe2x80x94dicyclohexylcarbodiimide
EDCxe2x80x94ethyldimethylaminopropylcarbodiimide
DDQxe2x80x94dichlorodicyanoquinone
HOAtxe2x80x941-hydroxy-7-azabenzoaminotriazole
THFxe2x80x94tetrahydrofuran
1*, 2* or 3* refers to once, twice or three times Burgess""s reagentxe2x80x94(methoxycarbonylsulfamoyl)triethylammonium hydroxide inner salt [Wipf, P.; Miller, C. P. J. Org. Chem. 1993, 58, 1575] Lawesson""s reagentxe2x80x942,4-bis(p-methoxyphenyl)-1,3-dithiaphosphetane=2,4-disulfide
The following are the preparations of compounds used to make the compounds of the invention in the examples which follow the preparations.
To a solution of salicylic acid (7 g, 0.05 mol) in methanol (100 mL) at xe2x88x9278xc2x0 C., SOCl2 (8 mL, 0.11 mol, 2.2 equiv.) was added dropwise. The reaction mixture was stirred at 40xc2x0 C. overnight, concentrated and extracted with EtOAc. The organic layer was washed with NaHCO3, brine, dried over Na2SO4, filtered and concentrated. The product was then dissolved in DMF; BnBr (9 mL, 0.075 mol, 1.5 equiv.), K2CO3(27.5 g, 0.2 mol, 4 equiv.) and NaI (75 mg) were added. The reaction mixture was stirred at room temperature for 12 h, diluted with EtOAc, washed with water, 0.5N HCl, and brine, dried over Na2SO4, filtered, concentrated and chromatographed on silica gel eluting with hexanes/EtOAc (10:1), to give methyl 2-(benzyloxy)benzoate as an oil (6.8 g, 0.03 mol, 57%). To the methyl ester was added KOH (3.2 g, 0.06 mol, 2 equiv.) and THF/H2O (1:1) (20 mL). The reaction mixture was stirred at room temperature overnight and extracted with EtOAc. The aqueous layer was acidified to pH 2 with 2N HCl and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated to afford 2-(benzyloxy)benzoic acid as white crystals (m=4.5 g, 70%).
Carbodiimide Method
To a stirred solution of 2-(benzyloxy)benzoic acid 3.53 g, 15.5 mmol) and L-serine benzyl ester hydrochloride (3.27 g, 14 mmol) in CH2Cl 2 (70 mL) was added Et3N (2.09 mL, 15 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.96 g, 15.5 mmol). After being stirred for 10 h at room temperature under argon, the reaction mixture was diluted with CH2Cl2 (200 mL), washed with H2O, saturated NaHCO3 solution, 5% citric acid aqueous solution, H2O, brine, dried over Na2S O4, filtered, and concentrated. Recrystallization from toluene afforded the title compound (5.12 g, 90%), as white crystals: Rf=0.41 (EtOAc/CH2Cl2=1/5); mp 116-118xc2x0; [xcex1]23D=+24.1xc2x0 (c=1.0, CH2Cl2); IR (KBr) 3500-3100, 1740, 1620 cmxe2x88x921; 1H NMR (300 MHz, CDCl3) xcex48.81 (d, J=6.9 Hz, 1H, NH), 8.19 (dd, J 1=7.8 Hz, J 2=1.8 Hz, 1H, ArH), 7.46-7.25 (m, 11H, ArH), 7.10-7.04 (m, 2H, ArH), 5.21-5.11 M, apparently 4 overlapping doublets, 4H, 2{CH2 phenyl}), 4.90-4.85 (m, 1H, CH), 3.93-3.89 (m,2H, CH2OH), 2.32 (br, 1H, OH); 13C NMR (75 MHz, CDCl3) xcex4170.13, 165.56, 156.91, 135.50, 135.26, 133.17, 132.31, 128.74, 128.53, 128.33, 128.06, 127.99, 121.48, 121.09, 112.79, 71,23, 67,17, 63.61, 55.38; FABMS: 406 (M+1). Anal. calcd. for C23H21NO5: C, 70.58; H, 5.41; N, 3.58, found: C, 70.43; H, 5.60; N, 3.37.
Active Ester-Carbodiimide Method
To a stirred solution of 2-(benzyloxy)benzoic acid (2.0 g, 8.7 mmol), L-serine benzyl ester hydrochloride (2.0 g, 8.7 mmol, 1 equiv.) and HOAt (1.2 g, 8.7 mmol, 1 equiv.) in DMF (200 mL) was added triethylamine (0.98 mL, 9.6 mmol, 1.1 equiv.) and EDC HCl (2.02 g, 4.8 mmol, 1.2 equiv.). After being stirred overnight at room temperature under nitrogen, the reaction mixture was diluted with ethyl acetate (200 mL), washed with 1N HCl (60 mL), saturated NaHCO3, saturated NH4Cl (60 mL), and brine (60 mL). The combined organic layers were dried over Na2SO4, filtered, concentrated and chromatographed on silica gel eluting with CH2Cl2/MeOH (95/5) to give the title compound, N-[2-(benzyloxy)benzoyl]-L-serine benzyl ester, 92.5 g, 70%) as white crystals. Rf=0.5 (CH2Cl2/MeOH, 95/5); 1H NMR (300 MHz, CDCl3) xcex48.80 (d, 1H, NH), 8.19 (dd,1H, ArH), 7.46-7.24 (m, 11H, ArH), 7.12-7.04 (m, 2H, ArH), 5.21-5.11 (m, 4H, 2{CH2phenyl}), 4.91-4.87 (m, 1H, CH), 3.93-3.90 (m, 2H, CH2OH), 2.2 (1H, OH).